Recent advances in genome research have provided new insight into the importance of membrane protein in all cells. In most eukaryotic organisms, over one-third of the open reading frames are predicted to be integral membrane proteins with anywhere from one to fourteen transmembrane segments. Membrane proteins provide many vital cellular functions including cell-cell communication such as recognition, adhesion, and membrane fusion; in material exchange including transportation and detoxication; and in processes of cellular energy conservation. High-resolution structural studies on a limited number of membrane proteins have contributed to our understanding of function of these biological macromolecules. The demand for structural knowledge of membrane proteins has increased more than ever in light of an increased number of membrane proteins for which important functions have been identified. However structural data on membrane proteins at atomic resolution is only being obtained rather slowly, mainly due to the tremendous difficulty in purifying sufficient quantity of membrane proteins, especially those of eukaryotic origin. There is a great need for improved methods of over-producing membrane protein and of producing diffraction quality membrane protein crystals. We, in collaboration with both intramural and extramural laboratories, explore the structure and function relationships of polytopic membrane proteins crystallographically by examining a few carefully selected membrane proteins. Our efforts are concentrated on those involved in cellular multidrug resistance (human P-glycoprotein) and respiration (cytochrome bc1complex of both mitochondria and bacteria). These studies have resulted in a better understanding of membrane protein architecture in general and of the mechanism of function of these important biological system in particular. It is hoped that our continued studies will aid in the development reagents with potential therapeutic value. Another area of interest is multicomponent protein complexes, such as those involved in regulated protein degradation. Cellular regulation requires protein remodeling activities, which affect intracellular protein degradation, folding, quality control, docking and interaction. One important aspect of protein remodeling is ATP-dependent protein degradation. Almost all important cytosolic and nuclear protein degradation is carried out by ATP dependent proteases, which often consist of a hexameric chaperone and either a hexameric or a heptameric protease and have been found in all organisms. The ClpAP protease, which we have begun to study crystallographically, is especially important for a number of reasons. First, ClpAP or its close homolog, ClpXP, are essential in many microorganisms. Second, ClpAP and ClpXP are highly conserved: ClpAP is found in the chloroplast of all plants and in photosynthetic bacteria, and ClpXP is found in the mitochondria of eukaryotes, including humans. Third, ClpA is the prototype of the Hsp100 molecular chaperones, which include yeast Hsp104, a chaperone shown to be involved in prion formation. Fourth, ClpA has significant sequence and structural similarity to AAA proteins, a broad class of protein conformation-transducing ATPases involved in a plethora of vital cellular functions. Last, Clp and other ATP-dependent proteases are structurally and mechanistically complex proteins, whose structure/function relationships reflect important biochemical principles that need to be understood at the sub-molecular level. We have determined the crystal structure of ClpA, the regulatory component of the ClpAP complex. ClpA consists of five tandemly connected structural domains corresponding to three functional groups. The N-terminal domain represents a novel fold of a repeating motif with a pseudo two-fold symmetry. The two AAA modules (D1 and D2) are connected head-to-tail with a 90? rotation. In the crystal, ClpA subunits form a hexameric spiral in which the D1-D1 interface has considerably more electrostatic contacts than the D2-D2 interface, providing structural basis for functional segregation of the two AAA modules. A symmetric hexameric ring model of ClpA locates a potential ClpP-interaction loop on the distal surface of D2 and reveals a large negatively charged central cavity. These studies will lead to a detailed understanding of sub-domains and specific amino acid side-chains involved in substrate recognition, ATP hydrolysis and the accompanying conformationa changes, interactions leading to assembly of the chaperone and the protease complex, and the mechanism of protein unfolding, translocation, and degradation carried out by this and similar essential regulatory chaperones.